It has been observed early life is an important time for the development of tolerogenic responses. This time is also correlated to the establishment of the intestinal microbiota, which is also necessary for the development of tolerance. However, it is unclear why tolerance is established best during early life and how the microbiota confers an effect on the immune system. Here I show gut resident bacteria spontaneously translocate from the intestinal lumen to the mesenteric lymph nodes during early life. This process occurs as part of normal development, yet the details of this phenomenon remain undefined. Adult mice can be manipulated through the use of antibiotics to induce bacterial translocation, which requires goblet-cell-associated antigen passages (GAPs) and CX3CR1+ antigen presenting cells (APCs). In this grant I will define when bacterial translocation occurs during early life, what species translocate, from where the species originate, and to which organs species translocate using 16S deep sequencing of murine organs throughout early life. I will also investigate if bacterial translocation during early life requires GAPs and CX3CR1+ APCs, similar to adult mice, and if manipulation of GAPs or CX3CR1+ APCs disrupt bacterial translocation using transgenic mice and pharmacological manipulation of GAPs. Finally, I will evaluate the role bacterial translocation has in the development of regulatory T cells and systemic tolerance using flow cytometry and a delayed type hypersensitivity model of systemic inflammation. Completion of this project will define the novel process of physiologic bacterial translocation during early lfe, how it is regulated and what effects it has on the immune system, providing insight into how the intestinal microbiota contributes to the development of tolerance and the effect disruption of bacterial translocation during early life has on lifelong immune responses.